NRL Memorandum Report 2324 ZInhibitors for Marine Sulfate-Reducing Bacteria in Shipboard Fuel Storage Tanks DOROTHEA E. KLEMME AND JOHN M. LEONARD Marine Biology and Biochemnistry Branch Ocean Scienres Division August 1971 Rp~d-cd bY NATIONAL TECHNICAL INFORMATION SERVICE Spr,hyfd Va. 22151 D D C r )F_7 hpn r7 pq SEP 23 1971 BLL U~jlrJ NAVAL RESEARCH LABORATORYC Washington, D.C. Approved for public release: distribution unlimited.
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NRL Memorandum Report 2324
ZInhibitors for Marine Sulfate-Reducing Bacteriain Shipboard Fuel Storage Tanks
DOROTHEA E. KLEMME AND JOHN M. LEONARD
Marine Biology and Biochemnistry BranchOcean Scienres Division
August 1971
Rp~d-cd bY
NATIONAL TECHNICALINFORMATION SERVICE
Spr,hyfd Va. 22151
D D C
r )F_7 hpn r7 pq
SEP 23 1971
BLL U~jlrJNAVAL RESEARCH LABORATORYC
Washington, D.C.
Approved for public release: distribution unlimited.
UNCLASSIFIEDSe-" un tv Classification
DOCUMENT CONTROL DATA -R & DfSvcurWy .1~tca~no title-, body .1 h,1tr1I_,i':d -- ti a fl*i-~ -- fs be ernteted whe~n the -w- all report 1, rl~j-1lied)
I SRIGINA TING ACTIVITY (Corpo rate author)a, REPORT ECURITY CLASSIFICATION
Naval Research Laboratory n gUNCLASSIIEDWashington, D.C. 20390 ab. nRme)
3 REPORT TITLE
INHIBITORS FOR MARINE SULFATE-REDUCING BACTERIA IN SHIPBOARDFUEL STORAGE TANKS
4 OESCRIPTIVE NO075 (Tpe of report and inclusive dates)
An interim report on a continuing problem.5 AUTHORiS) (First name, middle initial, lastrname)
Dorothea E. Klemme and John M. Leonard
i6 REPORT DATE 7a. TOTAL NO OF PAGES M1bI NO OF REFS
August 1971 22] 198&. CONTRACT OR GRANT N O 9a. ORIGINATOR'S REPORT NUMBER(S)
NRL Problem G04-01b. PBOJECT NO. NRL Memorandum Report 2324
RR -104 -03 -41-55039b. OTHER REPORT NO(S) (Any other numbers that may be assigned
I this report)
10. DISTRIBUTION STATEMENT
Approved for public release; distrition unlimited.
|11 SUPPLEMENTARY NOTES 112. SPONSORING MILITARY ACTIVITY
Department of the Navy(Naval Ship Systems Command)Washington, D.C. 20360
13. ABSTR ACTr
Problems of corrosive fuels, as well as corrosion of metals, from marine sulfate-
reducing bacteria in shipboard fuel tanks continue to plague the Navy. We have sought asolutionthrough finding a toxicant which can be added to the ballast or displacement waterand thus suppress the growth of the organisms. Despite their apparent hardiness in fueltanks, the bacteria have proven difficult to maintain in vigorous culture in the laboratory.So as a first step in bioassay, a system for maintaining an active inoculum was worked out,along with a test-tube procedure for estimating inhibitive dose. Fuels used in the assays todate are aviation gasoline and Navy Distillate. As candidate toxicants, seven materials wereselected for laboratory trial on the basis of prior toxicity information and presumed compat-ibility with the fuel. From the tube studies three thiopyridine derivatives were among thebest; experiments on a larger scale are suggested. Since the Navy foresees a wide use ofNavy Distillate, we should anticipate our problems by looking into its apparent microbialsusceptibility - and likewise susceptibility of Marine Diesel - to other classes of organisms.
12, Allred, R.C., "Methods Used for the Counting of Sulfate-ReducingBacteria and for the Screening of Bactericides", ibid, 22, No. 9,32-34 (1958)
13. Butlin, K.R., Adams, M.E., and Thomas, M. "The Isolation and Cultivationof Sulphate-Reducing Bacteria", J. gen. Microbiol. 3, 46-59 (1949)
14. Postgate, J.R., "Versatile Medium for the Enumeration of Sulfate-Reducing Bacteria", Appl. Microbiol. 11, 265-267 (1963)
11
15. Saich, A.M., Macpherson, R., and Miller, J.D.A., "The Effect ofInhibitors on Sulphate Reducing Bacteria: a Compilation", J. appi.Bacteriol. 27 (2), 281-293 (1964)
16. Guynes, G.J., and Bennett, E.G., "The Sensitivity of Sulfate-Reducing Bacteria to Antibacterial Agents. (The Mercurials)",Producers Monthly 22, No.11, 15-17 (1958)
17. Bennett, E.G., Guynes, G.J., and Isenberg, D.L., "The Sensitivityof Sulfate-Reducing Bacteria to Antibacterial Agents. (PhenolicCompounds)", ibid, 22, No.11, 18-19 (1958)
18. Bennett, E.G., Guynes, G.J. and Isenberg, D.L., "The Sensitivityof Sulfate-Reducing Bacteria to Antibacterial Agents ---l111, TheNitroparaffin Derivatives", ibid, 24, No.3, 26-27 (1960)
19. Sa1eh, A.M., "Differences in the Resistance of Sulphate-ReducingBacteria to Inhibitors", J. gen. Microbial. 37, 113-121 (1964).
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Table I. Toxicant Effects Upon Sulfate-heducing BacteriaUnder Two Distillate Fuels
Conc. Growth Responsel'ue____ Inhibitor ppm 3 days 10 days 21 days
- Arbitrary scale based on visible blackening when tube is shaken, viz.I slight gray, 4 = intense opaque black. All assays were run induplicate and since duplicate responses were always the same, a singlenumerical entry is shown in the table.
- DF-N was of Nigerian origin, DF-V of Venezuelan origin.
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Taole 2. Toxicant Effects Upon Sulfate-Reducing BacteriaUnder Avgas
(E) Sodium salt of dichlorophene 1* 1 4 420* -<I 1 1
(F) Hexahydro-i,3,5-tris- 5 4 4 4
(2-hyd roxyelhyl ) - 25 4 4 4
s-triazinej_/ 25 4 4 450 1 0 0
100 2 0 0
100 0 0 0
400 0 0 0(G) Methylene bisthiocyan/te-- 6 3 4 4
30 <1 0 0
60 0 0 0
Data were gathered in several experiments, each having growth controlscontaining no inhibitor. These controls were intense black throughout
except in the first experiment, designated by an asterick(*).I/ Arbitrary scale based on visible blackening when tube is shaken, viz.
1 = slight gray, 4 = intense opaque black. All assays were run induplicate and whenever the responses were the same, a single numericalrating is shown in the table; when the responses were different, both
ratings are given.
Concentrations given are based on active ingredient; material is 78.5%
pure.
3/ Concentrations given are based on active toxicant, which is 6% of the
product supplied.
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Table 3. Toxicant Effects Upon Sulfate-Reducing BacteriaUnder Navy Distillate Fuel
Data were gathered in several experiments, each having growth controls
containing no inhibitor. These controls were intense black throughoutexcept in the first experiment, designated by an asterisk(*).
1/-Arbitrary scale based on visible blackening when tube is shaken, viz.1 = slight gray, 4 = intense opaque black. All assays were run induplicate and whenever the responses were the same, a single numericalrating is shown in the table; when the responses were different, both
ratings are given.2/
-Concentrations given are based on active ingredient; material is 78.5%
pure.
-1oncentrations given are based on active toxicant, which is 6% of the
product supplied.
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APPENDIX
Formulas of Media
(1) API Vials: Difco Laboratories, Bacto-Sulfate API Broth, 9 ml per vial,prepared according to formulation given in the AmericanPetroleum Institute Recommended Practice 38, First Edition,1959, cited in Difco Supplementary Literature, Nov. 1966,
Magnesium sulfate 0.06 g Medium is dispensed intoAmmonium sulfate 0.30 g 20x125 mm screw-capped
Sodium sulfite 0.03 g tubes, 20 ml per tube,Dipotassium phosphate 0.06 g and autoclaved at 15 lb• *Ferrous ammonium sulfate 0.03 g for 15 min.; cooled toAscorbic acid 0.03 g about 500 C, then in-
Calcium lactate 1.05 g verted repeatedly untilDifco neopeptone 0.30 g medium gels.Difco agar 0.30 gSea water, filtered 100. ml
Trypticase 1.7 g Note - TSB without thePhytone 0.3 g dextrose, (BBL #11774)Sodium chloride 0.5 g is equally satisfactory.Dipotassium phosphate 0.25 gDextrose 0.25 g
For all TSB media in sea water, a 30% "stock" solution is prepared indistilled water and autoclaved at 15 lb for 15 minutes. The requiredvolume of this concentrate to give a 0.3% TSB can be added to membranefiltered sea water.
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! 1
(Page 18 is Blank)APPENDIX (cont'd)
TSB media as used in experiments:
(a) Basic TSB:
TSB, 30% stock solution 1.0 ml A 2.5% solution ofFerrous ammonium sulfate* 0.05 g Fe(NH 4) 2(SO4 ) 2.6H 20 isSea water, membrane filtered 97. ml prepared in distilled
water and 2 ml of thismembrane filtered (MF'd)into the sea water.
(b) Supplemented TSB:
TSB, LJ% stock solution 1.0 mlFerrous ammonium sulfate* 0.05 g in 2 ml dist. H 20, MF'dMagnesium sulfate 0.2 g 2in 5 ml dist. H0 , autoclavedSodium lactate, 60% 0.4 ml 2Ascorbic acid 0.01 g jin 2 ml dist. H0, MF'dSodium thioglycollate 0.01 g 2Sea water, membrane filtered 90. ml
*The Fe(NH 4) 2(SO 4) 2.6H 20 serves as indicator of production of H2 S
by the bacteria and hence as means of evaluating their growth.